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Using a serum-free defined medium, a human cell line, NCI-H929, was
established from a malignant effusion occuring in a patient with IgAk
myeloma. The cultured cells have the morphologic, ultrastructural,
biochemical, immunologic and cytochemical features of plasma cells. The
cells have rearranged alpha and kappa genes and synthesize and secrete
very high amounts of IgAk (>80 .mu.g/10.sup.6 cells/24 hr). The cells
express surface immunoglobulin (alpha and kappa), the plasma cell antigen
PCA-1, the transferrin receptor (T9) and T10, but lack antigens associated
with earlier stages of B cell development (HLA-DR, B1, B2, B4, CALLA), as
well as other leukocyte-macrophage antigens and Epstein-Barr virus nuclear
antigen. While the tumor cells were predominantly near-diploid, the
cultured cells are predominantly near-tetraploid with six copies of
chromosome 8, four to six of which have an 8q+ abnormality. The cultured
cells have a rearrangement of the cellular c-myc proto-oncogene (located
at 8q24) and express c-myc RNA.

Inventors:

Gazdar; Adi F. (Potomac, MD), Oie; Herbert K. (Rockville, MD)

Assignee:

The United States of America as represented by the Secretary of the
(Washington,
DC)

5. The cell line of claim 1 having a near tetraploid DNA content and chromosome number including six copies of chromosome 8, four to six of which have an 8 q+ abnormality.

Description

BACKGROUND
OF THE INVENTION

1. Technical Field

The present invention is related to the establishment and characterization of a new, cultured, human plasma cell myeloma line. More particularly, the present invention is related to a unique human plasma cell line, designated NCI-H929, having
rearranged in its genome the alpha and kappa genes and which is capable of synthesizing and secreting high amounts of IgAk (>80 .mu./10.sup.6 cells/24 hr), said cell line also having a rearranged cellular c-myc proto-oncogene.

2. State of the Art

Despite intense efforts in many laboratories during the past twenty years, plasma cell (PC) myelomas and leukemias have been one of the most difficult of human malignancies to establish in continuous culture (Karpas et al., Science 216:997,
1982). While a modest number of Holman & Stern, Chartered Folio P49604 `plasmacytoid` cell lines have been reported, the vast majority of these are lymphoblastoid cell lines (LCLs), which result from in vitro transformation of non-malignant B cells by
Epstein-Barr virus (Nilsson et al., Advan. Cancer Res. 37:319, 1982). In contrast, many mouse plasmacytoma and human Burkitt lymphoma (BL) lines exist (Marcu et al., Proc. Natl. Acad. Sci. USA 80:523, 1983; Taub et al., Cell 36:339, 1984). In
both of these otherwise very different B cell tumors, specific reciprocal translocations occur which bring the cellular myc proto-oncogene (c-myc gene) and an immunoglobulin (Ig) gene segment into the same chromosomal region. These events result in the
preferential transcription, and perhaps, abnormal regulation of the translocated c-myc gene. While similar molecular events probably occur in rat PC tumors (Sumegi et al., Nature 306:497, 1983), human myelomas have not been associated with rearrangement
or inappropriate transcription of c-myc, nor with structural abnormalities of its chromosomal location (8q24) (Philip, Cytogenet 2:79, 1980).

SUMMARY OF THE INVENTION

It is, therefore, an object of the present invention to establish in continuous culture a new cell line derived from malignant human plasma cells, said cell line being designated herein as NCI-H929.

It is a further object of the present invention to have a rearranged c-myc oncogene in said new plasma cell line.

Other objects and advantages will become evident upon a reading of the following detailed description of the present invention.

BRIEF DESCRIPTION OF DRAWINGS

These and other objects, features and many of the attendant advantages of the invention will be better understood upon a reading of the following detailed description when considered in connection with the accompanying drawings wherein:

FIG. 1 shows morphological appearances of NCI-H929 tumor and cultured cells. FIG. 1a, bone marrow aspirate at diagnosis. Almost all the cells are nucleated PCs, with occasional giant forms. Wright-Giemsa. FIG. 1b, histological section of
right pleura at autopsy. The cellular infiltrate consists predominantly of abnormal PCs with many hyperchromatic and bizarre forms. Hematoxylin and eosin. FIG. 1c, appearance of cultured cells. The very large cells are plasmacytoid, with prominent
nucleoli, occasional giant or multinucleated forms, and perinuclear clear areas (hof). Wright-Giemsa. FIG. 1d, immunocytochemical demonstration of kappa light chain. The staining is predominantly cytoplasmic with sparing of the hof areas.
Immunoperoxidase technique. All magnifications .times.300;

FIG. 2a and b show ultrastructure of NCI-H929 cell line. The appearance is that of an immature PC with large nucleus having a prominent nucleolus and clumped chromatin. The rough endoplasmic reticulum is extensive and arranged as perinuclear
lamellae (arrows). The golgi is well developed (arrowheads). Insert (FIG. 2b) is a higher magnification of the cytoplasmic features. Bars represent one .mu.m;

FIG. 3 shows DNA histograms of NCI-H929 malignant effusion and 91 day old culture. In both specimens all, or nearly all, of the cells are aneuploid. Of the tumor cells, 90% had a DNA index (DI) of 1.15 (peak 1), and 10% a DI of 2.3 (peak 2).
In the cell culture, peak 2 is the major component (77%) and peak 1 relatively small (8%). In addition, a third peak (peak 3) is present (15%), presumably representing the dividing (G.sub.2 and M) cells of peak 2;

FIG. 4 shows representative G-banded karyotype of NCI-H929 cells, 87 days after initiating culture. All six copies of chromosome #1 are abnormal; the two illustrated on the left have an apparent duplication of the long arm, while the other four
copies have deletions of the short arm of varying size. Of the six copies of chromosome #8, the one on the left is normal appearing; four and perhaps all five of the other copies have an 8q+ abnormality. Other consistent abnormalities included t(10q,
12p) in two of four copies, del (12p) in the other two copies of chromosome 12, and a marker chromosome (M1), possibly i(16q);

FIG. 5 shows Ig gene expression and rearrangement of NCI-H929 tumor and cell line. The figure consists of 12 lanes, which are numerically referred to in the legend sequentially from left to right.

Three DNA samples (lanes 1-3), a control T-cell line, NCI-H929 tumor and cell line, are shown in a BAM HI Southern genomic blot analysis probed with human alpha constant region-one. In all three DNAs the germline 14.5 kb band and in NCI-H929
Tumor and cell line a rearranged 13.0 kb band is identified (the 13.0 kb band is faint in NCI-H929 tumor). In lanes 4-6 analysis using the same probe is shown on three RNA samples, control (KK124, a mu and kappa producing Burkitt lymphoma cell line) and
NCI-H929 tumor and cell line synthesize a 1.9 kb Ig alpha mRNA. In lanes 7-9 three DNA samples, a control T-cell line and NCI-H929 tumor and cell line were digested with BAM HI restriction endonuclease and probed with human kappa constant region. All
three DNAs contain the germline 12.0 kb band and in NCI-H929 tumor and cell line a 14.5 kb rearranged band is identified. Lanes 10-12 present a Northern blot analysis using a kappa constant probe which identifies a 1.2 kb mRNA in control (KK124) and
NCI-H929 tumor and cell line RNA;

FIG. 6 demonstrates expression of surface antigens on PC line NCI-H929 and BL line St 486. NCI-H929 lacks expression of B1, B2 and HLA-DR; has weak expression of kappa light chain and strong expression of PCA-1. ST 486 lacks expression of B2
and PCA-1; has moderate expression of kappa light chain and very strong expression of HLA-DR. Because ST 486, an EBNA negative BL line, lacks expression of B2, an epitope of the common CR2 and EBV receptor, B2 expression by KK124, an EBNA positive BL
line, is demonstrated; and

FIG. 7 shows genomic blot analysis for c-myc in NCI-H929 tumor and cell line. Three DNA samples, control, NCI-H929 tumor and cell line are shown in an Eco RI Southern genomic blot analysis. The third exon of human c-myc probe identifies the
germline 12 kb Eco RI fragment in all three samples and an additional rearranged 14.5 kb band is present in the tumor and cell line. The relative intensity of the rearranged band is greater in the cell line than in the tumor sample.

DETAILED
DESCRIPTION OF INVENTION

The above and many other objects and advantages of the present invention are achieved by a human plasma cell myeloma line in continuous in vitro culture, said cell line having a rearranged c-myc proto-oncogene. Of course, the cell line can be
cryopreserved for convenient storage.

It is noted that all scientific and/or technical terms employed herein have the same meaning as generally understood by one of ordinary skill in the art to which the invention belongs. All publications cited hereunder are incorporated herein by
reference. Although any similar or equivalent methods and materials may be employed in the practice, testing and the like of the present invention, the preferred methods and materials are now described.

The term "rearranged" as used herein means a pattern of restriction endonuclease sites that deviate from the normal human germ line DNA.

Described herein is the establishment of a cell line, NCI-H929, from a malignant effusion occurring in a patient with IgAk myeloma. The patient's tumor and cultured cells have the morphologic and antigenic properties of fully differentiated
malignant PCs, lack EBV nuclear antigen (EBNA), and secrete very high concentrations of IgAk (>80.mu.g/10.sup.6 cells/24 hr). Of major significance is the finding that a rearrangement of the c-myc oncogene is present in both the tumor and cultured
cells, in association with an addition to the long arm of chromosome 8 (8q+).

MATERIALS AND METHODS

A 62 year old white woman was diagnosed as having IgAk PC myeloma. Her bone marrow was virtually replaced with immature PCs (FIG. 1a), and she had a serum IgA level of 9 g/dl. She was treated with vincristine, melphalan, cytoxan and prednisone,
and achieved a brief clinical response. At relapse, nine months after diagnosis, a right pleural effusion was processed for diagnostic workup. The patient died a month later and autopsy revealed widespread infiltration of the viscera, including lungs
and pleura, with malignant PCs, many of which had bizarre or multinucleated forms (FIG. 1b).

Cell culture and characterization.

The NCI-H929 cell line was initiated from the malignant pleural effusion from the patient with IgAk myeloma. The cells at the interface of a discontinuous Ficoll-Hypaque gradient were harvested, washed, resuspended in various media in 75
cm.sup.2 flasks at density of about 1.times.10.sup.6 /ml and incubated in a humidified 6% CO.sub.2 atmosphere at 37.degree. C. Cytological examination of the interface cells indicated that over 90% consisted of malignant PCs. The media used included
RPMI-1640 supplemented with 10% fetal bovine serum or 10-20% patient's pleural fluid, or serum-free media capable of supporting continuous growth of the NCI-H929 cell line. An example of such a medium is an ACL-3 medium comprising RPMI-1640 medium
supplemented with insulin (20 .mu.g/ml), transferrin (10 .mu.g/ml), sodium selenite (2.5.times.10.sup.-8 M), hydrocortisone (5.times.10.sup.-8 M), epidermal growth factor (1 ng/ml), ethanolamine/phosphorylethanolamine (1.times.10.sup.-5 M of each),
triiodothyronine (1.times.10.sup.-10 M), bovine albumin (0.2%), sodium pyruvate (0.5 mM), HEPES buffer (10 mM) and extra glutamine (2 mM). Mycoplasma contamintion was tested by routine liquid and agar plate culture techniques well known in the art and
by the Hoechst stain method by Microbiological Associates, Bethesda, MD. Species identification was by isoenzyme analysis using agarose electrophoresis (Authentikit apparatus and reagents, Corning Medical, Medfield, MA.).

Morphologic studies.

Air dried cytospins of cultured cells were stained with Wright-Giemsa or other suitable stains. For transmission electron microscopy, cell pellets were fixed in 2.5% glutaraldehyde, post-fixed in chrome-osmium and stained with uranyl acetate
following routine procedure. Thin sections were examined in a Siemens Elmiskop 1A electron microscope.

It was found that no significant growth occurred in media supplemented with bovine serum or clarified effusion fluid. In contrast, immediate and continuous growth of cell line NCI-H929 occurred in fully defined ACL-3 medium, supra. The cultured
cells have a population doubling time of about 50 hr, and grow as irregularly shaped floating cells, either singly or in small, loose clusters. Giant forms are frequent, some of which are multinucleated. Cell size varies from 20 to over 50 .mu.. The
cell line is free of mycoplasma contamintion.

A deposit of the cultured NCI-H929 has been made at the American Type Culture Collection, Rockville, MD. under the accession number CRL 9068. Upon issuance of a patent, the deposit will continue to be viably maintained for at least 30 years and
will be available to the public without restriction, of course, in accordance with the provisions of the law.

DNA histograms (FIG. 3) of the gradient concentrated pleural fluid cells indicated that almost all the cells were aneuploid, with 90% having a DNA index (DI) of 1.15 (peak 1), and 10% a DI of 2.3 (peak 2). After culture, peak 2 became the
dominant population (FIG. 3). Cytogenetic tests of the cell line at day 87 of culture (FIG. 4), indicated that approximately 80% of the metaphases consisted of near tetraploid cells (90-92 chromosomes) with a minor near diploid subpopulation (44-48
chromosomes). Banding studies demonstrated a human karyotype with multiple structural abnormalities (FIG. 4). Of particular significance was the finding that six copies of two chromosomes (numbers 1 and 8) were present in the near tetraploid cells.
Additional chromosomal material of unidentified origin was attached to the long arm of chromosome 8 (8q+), in at least four and usually in all six copies. All six copies of chromosome 1 were abnormal; one pair demonstrating an apparent duplication of
the long arm, dup(1) (q11-25); the other 4 having a delection of the short arm of variable length.

Ig and antigen studies.

Immunodiffusion studies demonstrated that the pleural fluid, culture medium and extracts of cultured cells contained IgAk. The presence of intracytoplasmic Ig was confirmed by immunoperoxidase stain (FIG. 1d). Quantitative studies demonstrated
that the cultured cells were secreting about 83 .mu.g/10.sup.6 cells/24 hr and contained 7.1 .mu.g/10.sup.6 cells of intracytoplasmic IgA. These high concentrations were confirmed by an ELISA method. DNA and RNA from the tumor and cell lines were
analyzed with .sup.32 P-labelled probes derived from the Ig constant regions (FIG. 5). Patterns from both samples were the same: one germline and one identically rearranged alpha and kappa genes were noted, while both mu genes were deleted. Abundant
alpha and kappa mRNA were present, confirming the IG production data (FIG. 5).

Flow cytometric studes of both tumor and cultured cells demonstrated the presence of alpha and kappa surface immunoglobulin (2+), PCA-1 (3+) and T10 (2+) antigens, and absence of other B cell antigens (B1, B2, B4, CALLA, I2 and HLA-DR).
Representative antigen expression patterns are illustrated in FIG. 6. In addition, the following lymphocyte and macrophage associated antigens also were absent: L1, L2, L3, L4, L7, L8, 19, L11, L12, LeumM1, LeuM3 and TAC. The cells lacked expression of
terminal deoxynucleotidyl transferase and EBNA. The transferrin receptor (T9 antigen) was not detected on the tumor cells, but was present on the cultured cells.

The antigen expression patterns of other B cell lines were compared to those of NCI-H929 (Table 1). The B lymphoblastoid and BL lines always expressed very high concentrations of HLA-DR, consistent expression of B1 and B4, and occasional
expression of B2, CALLA and T10. In addition, some lines expressed low levels of PCA-1. EBNA was present in all LCL and some BL lines. The "plasmacytoid" lines demonstrated considerable heterogeneity. The HS Sultan and ARH 77 lines were EBNA positive
and their surface antigen patterns indistinguishable from those of LCLs. U266 cells were EBNA negative and expressed PCA-1 and HLA-DR, but lacked other B antigens. RPMI 8226 lacked EBNA and HLA-DR, but expressed moderate amounts of T10 and PCA-1 and
low amounts of B1 and B2.

The state of the c-myc gene in the patient's tumor and cultured cells were examined. This analysis (FIG. 7) revealed germline and rearranged bands (12 and 14.5 kb, repectively) in both the tumor and the cell line, demonstrating that a DNA
rearrangement had occurred near the c-myc gene. In the tumor cells the germline band was of greater intensity than the rearranged one, while in the cultured cells both bands were of approximately the same intensity. To determine whether the rearranged
band reflected an Eco RI restriction enzyme polymorphism, the DNA samples were digested with the restriction enzymes Hind III, Sst I and Xba I. In all cases a rearranged band was seen (data not shown), ruling out a simple restriction site polymorphism.
c-myc RNA was present in both the tumor and cultured cells as determined by Northern analysis (data not shown).

In summary, the results presented herein clearly demonstrate the establishment, using serum-free defined medium, of a human PC culture. The cell line has the characteristic morphology, ultrastructure and cytochemical features of PCs. It
secretes very high concentrations of the same Ig class (IgAk) as did the patient's tumor cells, more than ten-fold higher than those reported for other human lines. Identical Ig gene rearrangements were detected in the tumor and cultured cells,
confirming that both were derived from the same clone of malignant B cells and provide a molecular signature for the cell line.

To determine the relationship of NCI-H929 cells and other human "plasmacytoid" lines to LCL and BL lines, both of which may secrete Ig, the surface expression patterns of B cell specific and associated antigens were compared. Most normal and
malignant B cells, other than PCs, express B1, B4 and Ia-like antigens (HLA-DR and I2), while B2 and CALLA have more stage restricted expression patterns. PCs and myeloma cells lack expression of these antigens, but acquire expression of a plasma cell
associated antigen (PCA-1) and T10, an antigen present on various activated lymphocytes. PCs and myeloma cells lack receptors for EBV (B2 antigen) and are EBNA negative. The antigen expression patterns of NCI-H929 tumor and cultured cells were
identical, except for the absence of the transferrin receptor, T9, on the former. Myeloma tumors have a low proliferative component. Expression of T9 is associated with dividing cells, including mouse myeloma cells, and it is present on the cultured
cells which have a much higher proliferative fraction than the tumor cells. Both the tumor and cultured cells had antigen patterns characteristic of fully mature PCs, and could readily be distinguished from LCL and BL lines.

NCI-H929 tumor and cultured cells demonstrated an identical rearrangement of the c-myc gene. The rearranged DNA fragment was readily observable on Eco RI digests of genomic DNA, and its concentration increased relative to the germline band
during culture. The use of other restriction enzyme digests excluded the possibility that the DNA rearrangement was due to a simple Eco RI restriction enzyme site polymorphism. Without being bound to any theory, it is postulated that the relative
increase of the rearranged band during culture could be due to: (a) loss of contaminating stromal cells during culture; (b) selective growth of a tumor cell subpopulation having the rearrangement; or (c) selective increase in the copy number of the
rearranged gene. DNA index studies excluded significant contamination of the tumor preparation with stromal cells. The tumor cells consisted predominantly of near diploid cells with a minor neartetraploid subpopulation. During culture, selective
growth of the latter subpopulation occurred. The near tetraploid cells had 6 copies of chromosome 8, and 4-6 of these had an 8 q+ abnormality. In BL a reciprocal translocation occurs between the long arm of a single copy of chromosome 8 near the site
of the c-myc gene (8q24) and one of the Ig loci (kapa, 2p11; mu, 14q32; lambda, 22q11). In NCI-H929 cells, no structural abnormality of chromosomes 2, 14 or 22 was apparent.

It is noted that the use of a serum free defined medium was critical for culture establishment, as the tumor cells initially failed to proliferate in serum containing media.

NCI-H929 is a highly differentiated human PC line. Antigenic and molecular studies confirm that it is derived from the patient's myeloma cells. It is an important addition to a very small, select number of human lines. It is unique in the
degree of its differentiation and quantity of Ig secreted. In addition to being a potential fusion partner for human monoclonal antibody production, the culture offers a model for studying terminal B cell differentiation, Ig secretion and the
relationship between human myeloma cells and the c-myc gene.

It is understood that the examples and embodiments described herein are for illustrative purposes only and that various modifications or changes in light thereof will be suggested to persons skilled in the art and are to be included within the
spirit and purview of this application and the scope of the appended claims.